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Improved Genome Editing Efficiency and Flexibility Using Modified Oligonucleotides with TALEN and CRISPR-Cas9
Jean-Baptiste Renaud1, Charlotte Boix1, Marine Charpentier1
1INSERM U1154, CNRS UMR7196, Museum National d'Histoire Naturelle, Paris 75005, France.
Cell Reports
|March 1, 2016
Summary
Phosphorothioate-modified oligonucleotides significantly boost genome editing efficiency for precise DNA insertions. This method allows for larger insertions and high-frequency editing in vivo, overcoming previous limitations.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Genome editing technologies like CRISPR-Cas9 and TALEN enable precise DNA modifications.
- Homology-directed repair (HDR) is used for sequence editing but often has lower efficiency than gene knockout.
- Optimal strategies for efficient HDR-mediated editing with donor DNA are not well-established.
Purpose of the Study:
- To investigate methods for enhancing the efficiency of homology-directed repair in genome editing.
- To evaluate the utility of phosphorothioate-modified oligonucleotides as donor DNA for precise genome editing.
- To assess the feasibility of this enhanced editing strategy in various cell types and animal models.
Main Methods:
- Utilizing CRISPR-Cas9 and TALEN nucleases for genome editing.
- Employing single-stranded oligonucleotide (ssODN) donors modified with phosphorothioate linkages.
- Testing editing efficiency in cultured cells and in vivo in rats and mice.
- Analyzing insertion accuracy and frequency, including homozygous loxP site insertion at the mouse ROSA locus.
Main Results:
- Phosphorothioate-modified ssODN donors markedly enhance genome editing efficiency compared to unmodified donors.
- This approach allows for greater design flexibility, accommodating insertions exceeding 100 bp.
- High-frequency targeted sequence insertions were successfully achieved in rats and mice, with no significant toxicity observed.
- Precise homozygous loxP site insertion was demonstrated at the mouse ROSA locus.
Conclusions:
- Phosphorothioate modification of oligonucleotide donors is a powerful strategy to improve HDR-mediated genome editing efficiency.
- This method offers a robust and flexible approach for generating precise genetic modifications in various systems.
- The high efficiency and successful application in vivo pave the way for advanced genome engineering applications.
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